| Tumor stem cells (cancer stem cells) are a small subset of the tumor cell population with stem cell properties. They possess the potential for self-renewal. They are the cause of the formation of various degrees of differentiation seen in tumor cells and tumor expansion. Cancer stem cells have been successfully isolated from hematopoietic malignancies, breast cancer and brain tumor tissue. This proves the existence of stem cells in the tumor cell population can cause groups of tumor to amplify.Tumor cells possess the characteristic of heterogeneity. Tumor cells from one clone can form subclones that differ in their growth process, their propensity to invade, growth rate, degree of differentiation, their hormone response and their variable sensitivity to anti-cancer drugs. The theory of cancer stem cells can explain the phenomenon of tumor cell heterogeneity, i.e., cancer stem cells differentiate in different directions based on function, they mature under different selection pressures resulting in the drift of tumor cell groups, thus resulting in heterogeneity. Therefore, since cancer stem cells are the source of the tumors, the eradication of cancer stem cells means the elimination of the tumor.Biological characteristics of cancer stem cells (CSC/TSC)1Self-renewal:the growth and proliferation of the tumor is guided by the tumor stem cells, cancer stem cells similar to adult stem cells that also have the characteristics of self-renewal and maintain the continued growth of the tumor through self-renewal. Another important reason for tumor growth is that an increasing number of tumor cells have self-renewal capacity caused by disorders in certain genes involved in the regulation mechanism in stem cells. In addition, several important signaling pathways that regulate normal self-renewal of stem cell including Notch and Wnt, NF-kB and Shh also play an important role in self-renewal in tumor stem cells.The proliferation of normal stem cells display self-stability in vivo. Their number in the proliferation process is maintained in a constant state. Cancer stem cells do not have self-stability and errors in gene duplication during proliferation cannot be repaired2Differentiation potential:tumor stem cells originate from mature blocked normal stem cells and have the ability to generate progeny of different degrees of differentiation of cancer cells and form new tumors in vivo. Tumor cell differentiation is different in the same tumor tissue, where the degree of malignancy of tumor cells is low in mature differentiation, while poorly differentiated tumor cells are highly malignant.3High tumorigenicity:a large number of trials have proved cancer stem cells have higher tumorigenic potential than non-tumor stem cells. Beier et al reported that in nude mice inoculated with22kinds of malignant glioma cells,11kinds containing CD133+cancer stem cell populations had significant growth and tumorigenicity,4kinds with CD133-cell populations grew into tumors slowly, while seven malignant glioma cell-derived cells did not grow. Singh et al reported that each mouse inoculated with100CD133+cancer stem cells resulted in the formation of a tumor6months after inoculation; at the same time the inoculation of100000CD133-non-tumor stem cells in mice did not form tumors. The results above support that cancer stem cells have a higher tumorigenic potential than non-tumor stem cells.4Drug resistance:drug resistance is one of the characteristics of tumor stem cells. Many reasons cause tumor treatment failure, multidrug resistance (mutidrug, resistance, MDR) being one of the main reasons. The membrane of cancer stem cells express the proteins of the ABC transporter family that can transport and discharge metabolites, drugs, toxic substances, peptides, nucleotides and other substances, thus significantly decreasing the effectiveness of many chemotherapeutic drugs in inhibiting or killing tumor stem cells.It has long been known that hypoxia in solid tumours is related to poor prognosis. Traditional chemotherapy is aimed at rapidly proliferating tumor cells. Proliferation of TSC in cell populations is slow, and thus insensitive to chemotherapy. TSC membrane protein regulated by HIFs, can be efficiently excluded from the drug in a hypoxic environment. TSC in a hypoxic environment can be better protected. Hypoxic cells are not sensitive to radiotherapy, hypoxia being a general feature of glioblastoma. Studies have shown that the radiation resistance of glioblastoma stem cells is attributed to the enhancement of DNA repair capacity suggesting that radiation resistance of stem cells will significantly improve under anaerobic conditions. Therefore, hypoxia is an important target for cancer treatment whether chemotherapy or radiotherapy. Clinical observations reveal that TSC treatment-resistance is the reason why tumors are difficult to eradicate. Clinical treatment targeting TSCs have received extensive attention. A recent study found that in brain tumor stem cells associated with tumor microvasculature, the TSC distribution and positioning of endothelial cells are closely related. Endothelial cells provide oxygen for the TSC. Neuroblastoma cell lines express of HIF-2a under5%O2. Neuroblastoma tissue rich in blood vessels still have significantly high HIF-2a expression that indicates that in vivo tumors rich in blood vessels still remain in hypoxic states possibly because of malformed vasculature. This view suggests that tumor microvascularized areas are likely to be the TSC niche. Targeting these vascular niches can more effectively remove the TSC.MicroRNAs (miRNAs) are a group of single-stranded RNA (～22nt) encoded by the animals, plants, and the viral genome. They do not have an open reading frame (ORF) and do not encode protein but participate in a variety of important physiological and pathological processes in the body. They are able to target mRNA3-UTR of complementary base pairing (untranslated region) area. Thus, degradation or inhibiting its expression by silencing specific genes that regulate body growth, development and diseases especially has an important regulatory function on tumor occurrence and development. In recent years, miRNA has become a hot research field of molecular biology, genetics and clinical medicine. About one-third of the human gene encoding mRNA is presumably negatively regulated by microRNA.MiRNA has the following characteristics:①specificity:different organizations of different cells have specific miRNA expression patterns and sequence features, which can be used as specific molecular markers of certain tissues or cells.②Sequential:miRNA composition is different at different developmental stages. Specific miRNA in a specific stage of specific cells determine the direction of cell differentiation and the differentiation phase, regulate the timing and direct the differentiation switch. For example miR-3to miR-7genes expressed in Drosophila in early embryogenesis, are not expressed in other phases. Drosophila begins to express miR-12, miR-21and miR-28from its larval stage, and these are maintained at a high level in the adult stage.③conservative:Different species, tissues, organs and cells with the same or similar miRNA molecules have similar regulatory functions. Three individual human embryonic cells miRNA (miR-302b, miR-302c, miR-302d) and mouse embryonic cells are same.miR-371and miR-302family is the same in both humans and mice.④miRNA targets:Mainly are time and space-specific transcriptional regulation genes and apoptosis regulatory genes. They also regulate specialized cell function and structure mediated through the balance between cell proliferation and apoptosis.Abnormal miRNA expression is found in a variety of human tumors,some miRNA specific expression is found in stem cells. High expression of miRNA-17and miRNA-92in human lung cancer and B-cell lymphoma can promote tumor cell proliferation. Chen et al found that content of some complex miRNA in embryonic stem cells is less in the mature somatic tissues. A large number of known tumorigenic miRNAs expressed in some of the original cells but its expression gradually decreased with cell differentiation. Silber et al found miR-124and miR-137had a low expression in malignant gliomas but had a high expression in the differentiated mouse neural stem cells. These two types of miRNA over-expression in glioma oligodendrocytes and malignant glioma stem cells in mice can lead to morphological changes and loss of self-renewal and tumorigenicity. Scholars have proposed that the degree of differentiation of cells or tissues can be detected by specific miRNA markers and there is evidence showing that non-coding miRNA expression distinguishes between stem cells and differentiated mature cells. The important role of miRNA in cancer and some of the miRNA-specific expression in stem cells provide evidence that these miRNA help in the transformation from stem cells to malignant cells.Spectrum changes and the identification of target mRNA through miRNA expression provides a new approach for cancer treatment strategy.There are several ways:they may have a carcinogenic effect through inhibition of miRNA; degradation of cancer-causing mRNA through complementary competition;3stimulate miRNA with a tumor suppressor role;4.external modification of miRNA expression. Although we know very little about the biological function of miRNA in tumorigenesis, and their relationship is poorly understood, people have begun to explore how miRNA may be applied in tumor treatment.Cheng et al inhibited human miRNA by designing antisense oligonucleotides to interfere with the role of miRNA in cell growth and apoptosis. The synthetic antisense oligonucleotide complementary miRNA oncogene known as the anti-miRNA oligonucleotides (anti-miRNA oligonucleotide AMO) can effectively inhibit the activation of carcinogenic miRNA in tumors thereby delaying tumor growth. Inhibition of miRNA activity through successively administered specifically designed antisense oligonucleotides miRNA, such as of miRNA-155antisense oligonucleotides is more stable and less toxic than other cancer treatment method.AMO and cholesterol-conjugated anti-miRNA oligonucleotides (antagomirs) inhibit miRNA activity after injection in mice in various organs. Several studies have shown that the breakthrough achieved in the modified miRNA expression at some biological systems. By using miRNA, perfectly complementary antisense oligonucleotides can be designed for the specific downregulation of miRNA expression of the nucleotide sequence. This oligonucleotide is called an antagomir. Recently, antagomir treatment of cancer was proven effective. Tumor growth of neuroblastoma in nude mice can be aborted by antagomir-17-5p. Another more effective method is antisense oligonucleotides that play a similar miRNA sponge role.These sponges from the transgenic RNA, by a strong and specific miRNA competitive complementary combination leads to disinhibition of targets. The transfection of exogenous miRNA-agomir complementary to a specific cancer-causing mRNA can result in the corresponding mRNA inactivation. Stimulating tumor inhibiting miRNA provides another theoretical basis for the treatment of tumors. For example, in order to achieve the over-expression of let-7we transfect let-7on lung cancer cells resulting in inhibition of tumor cell growth.miRNA can affect the function of external mechanisms, and external mechanisms affect miRNA expression. Therefore, proteins such as DNA methyltransferase, group deacetylase inhibitor and drugs can affect miRNA expression. For example, methylation of the CpG site promoter and histone deacetylation lead to downregulation of miRNA-127levels in bladder cancer cells. Using these two kinds of drugs in combination can reduce DNA methylation and histone acetylation resulting increasing miRNA-127levels.miRNA-127plays a tumor suppressor function by blocking cell proliferation. All of these treatment strategies are based on the miRNA targeting.miRNA therapy or a combination of treatment with traditional medicines has good prospects. Due to the relationship between a variety of cellular functions and miRNA, their roles in various signaling pathways are still in research. This miRNA-related technology has not yet entered clinical application.MiRNA plays an important role in tumorigenesis, development, invasion and metastasis. A lot of achievements have been made in related fields that have laid the foundation for the use of miRNAs in tumor diagnosis and treatment. Imbalance in mechanisms of MiRNA function in tumor expression still needs further study. With further research the relationship between miRNA and cancer will be clarified and the application of miRNA in cancer therapy will also have a broader outlook and become a new strategy for cancer treatment.This study is divided into the following three parts:Part I:(1)U87cells were seeded in humidified incubator in DMEM/F12medium containing10%FBS at37℃,5%CO2saturated humidity in incubator culture. Passage after5-7d according to the ratio of1:2or1:3.(2)CSCs culture and subculture:U87cells were inoculated in FBS-free DMEM/F12medium with B27, heparin, epidermal growth factor, basic fibroblast growth factor at37℃,5%CO2. The supernatants (containing ball of cells) were drawn4days after U87cells form ball of cells and then blown into a single cell suspension and passaged according to the ratio of1:2or1:3.(3)CSCs identification:The cell balls were collected when the original generation cells forming the ball had reached100-200cells, fixed with2%poly-formaldehyde for15min at room temperature;10%donkey serum for10min, add mouse anti-human CD133,4℃for overnight; Cy3-labeled rabbit anti-mouse secondary antibody was added and incubated at room temperature for2h, Hoechst33342dye nuclear. Positive CSCs count was conducted of randomly selected20fields of vision under a microscope.(4)Cells were placed in the hypoxic state culture using the hypoxic cell incubator, the culture conditions were37℃,5%CO2,1%O2. Application of microRNA chip system was used to study miRNAs of U87tumor stem cells and their relation to hypoxia.Part II:Application of real-time fluorescent quantitative RT-PCR method on four miRNA (hsa-miR-124hsa-miR-143hsa-let-7i hsa-miR-29c) and verification. This experiment used the total RNA of hypoxic and normal U87tumor stem cell as a template, using a two-step quantitative PCR to amplify the target gene.U6rRNA was used as an internal standard.2-ΔΔCt method was used to calculate the relative quantitative value of each miRNA in different phases after the reaction.(Chip results:hsa-miR-124up11.79441663times, hsa-miR-143raised4.748774253times,hsa-let-7i down2.087019times,hsa-miR-29c down2.0929982times).That consistent with the microarray results, verifying the reliability the microarray results.Part III:Tumor stem cells were taken while in the logarithmic phase U87and counted. They were resuspended without antibiotic medium, inoculated with3×105cells per well in6well plate, medium added to2ml16-24h after transfection.(1) The specificity of synthesis of miRNA.(miRNA oligonucleotide mimics), transfection of human U87tumor stem cells and RT-PCR detection of hsa-let-7i expression levels were carried out;(2) Western Blot. Detection of Bcl-2, Caspase3, Caspase9protein’s expression in U87tumor stem cells was carried out (3) TUNEL detection of U87tumor stem cells apoptosis(4) flow cytometry detection of miRNA mimics apoptosis induction.Results:(1) After Transfection of hsa-let-7i miRNA levels of U87MG cells significantly increased.(2).Western blot results showed that: compared with the control group, Bcl-2protein levels was significantly reduced, Caspase3and Caspase9protein levels were significantly increased48hours after transfection of U87tumor stem cells.(3)The U87tumor stem cells showed apoptosis compared with control group by flow cytometry.(4).TUNEL detected the U87tumor stem cells, results showed that markers have enhanced fluorescence in apoptotic cell after increase of hsa-let-7i expression levels. |
PDF Full Text Request